Design of wogonin-inspired selective cyclin-dependent kinase 9 (CDK9) inhibitors with potent in vitro and in vivo antitumor activity

Article abstract of DOI:10.1016/j.ejmech.2019.06.024

Wogonin, a natural product isolated from the plant Scutellaria baicalensis, has been shown to be a potent and selective inhibitor of CDK9. With the purpose of investigating the activity and selectivity of this chemical scaffold, several series of wogonin derivatives were prepared and screened for CDK9 inhibition and cellular antiproliferative activity. Among these compounds, the drug-like compound 51 showed potent activity against CDK9 (IC₅₀ = 19.9 nM) and MV4-11 cell growth (IC₅₀ = 20 nM). In addition, compound 51 showed much improved physicochemical properties, such as water solubility, compared with the parent compound wogonin. The follow-up studies showed that the compound 51 is selective toward CDK9-overexpressing cancer cells over normal cells. Preliminary mechanism studies on the anticancer effect indicated that 51 inhibited the proliferation of MV4-11 cells via caspase-dependent apoptosis. In addition, highlighted compound 51 showed significant antitumor activity in mouse acute myeloid leukemia (AML) models without producing apparent toxic effects in vivo, which gave us a new tool for further investigation of CDK9-targeted inhibitor as a potential antitumor drug especially for AML.

Full text of DOI:10.1016/j.ejmech.2019.06.024

Accepted Manuscript
Design of wogonin-inspired selective cyclin-dependent kinase 9 (CDK9) inhibitors with
potent in vitro and in vivo antitumor activity
Jubo Wang, Tinghan Li, Tengteng Zhao, Tizhi Wu, Hong Ding, Zhiyu Li, Jinlei Bian
PII:
S0223-5234(19)30549-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.06.024
EJMECH 11427
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2019
Revised Date: 28 May 2019
Accepted Date: 8 June 2019
Please cite this article as: J. Wang, T. Li, T. Zhao, T. Wu, H. Ding, Z. Li, J. Bian, Design of wogonin-
inspired selective cyclin-dependent kinase 9 (CDK9) inhibitors with potent in vitro and in vivo
antitumor activity, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/
j.ejmech.2019.06.024.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design of wogonin-inspired selective cyclin-dependent kinase 9 (CDK9)
inhibitors with potent in vitro and in vivo antitumor activity
Jubo Wang a,1, Tinghan Li a,1, Tengteng Zhao a, Tizhi Wu a, Hong Dingb, Zhiyu Li a,*, Jinlei Bian a,
*
b
aJiangsu Key Laboratory of Drug Design and Optimization, Department of Medicinal Chemistry, China
Pharmaceutical University, Nanjing, 211198, China.
b State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences.
1 These two authors contributed equally to this work.
*Corresponding author. E-mail: zhiyuli@cpu.edu.cn (Z, Li), bianjl@cpu.edu.cn (J. Bian)
Abstract
:
Wogonin, a natural product isolated from the plant Scutellaria baicalensis, has been shown to be a
potent and selective inhibitor of CDK9. With the purpose of investigating the activity and selectivity of
this chemical scaffold, several series of wogonin derivatives were prepared and screened for CDK9
inhibition and cellular antiproliferative activity. Among these compounds, the drug-like compound 51
showed potent activity against CDK9 (IC50 = 19.9 nM) and MV4-11 cell growth (IC50 = 20 nM). In
addition, compound 51 showed much improved physicochemical properties, such as water solubility,
compared with the parent compound wogonin. The follow-up studies showed that the compound 51 is
selective toward CDK9-overexpressing cancer cells over normal cells. Preliminary mechanism studies
on the anticancer effect indicated that 51 inhibited the proliferation of MV4-11 cells via
caspase-dependent apoptosis. In addition, highlighted compound 51 showed significant antitumor
activity in mouse acute myeloid leukemia (AML) models without producing apparent toxic effects in
vivo, which gave us a new tool for further investigation of CDK9-targeted inhibitor as a potential
antitumor drug especially for AML.
Keywords: Wogonin, CDK9, AML, flavonoid, antitumor

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1. Introduction
Cyclin-dependent kinases (CDKs) are members of the serine/threonine kinase family, and act as
heterodimeric complexes including a catalytic subunit and a regulatory subunit called cyclin. CDKs are
generally divided into cell-cycle CDKs and transcriptional CDKs, depending on their specific roles
[1-2]. Given their central roles in cell-cycle progression, transcription and many other biological
processes, CDKs together with their corresponding cyclins have been pursued as logical targets for
anticancer therapy for more than a decade, and a large number of CDK inhibitors have been developed
as potential anticancer drugs [1-3]. The first generation of CDK inhibitors, such as flavopiridol,
R-Roscovitine and SCH 727965 (Fig. 1), were almost non-selective with marked inhibitory activity and
able to inhibit CDKs at nanomolar concentrations [4]. However, early efforts to find pan-CDKs
inhibitors led to a narrow therapeutic window and serious side effects, which limits their clinical
advantages [5].
Numerous studies have found that tumor cells lacking CDK1/2/4/6 can still proliferate normally
and CDK2/4/6 knockout mice remain viable, which suggests a highly functional redundancy and
compensatory mechanisms among CDK family members, especially cell-cycle CDKs [6–8].
Accordingly, inhibition of transcriptional CDKs has gained increasing attention in recent years
following the observation that most pan-CDKs inhibitors exert their antitumor activity through the
CDK9-mediated down-regulation of transcription of anti-apoptotic proteins such as MCL-1 [9–12].
The overexpression of CDK9 can promote the expression of MCL-1, which is involved in the
pathogenesis of acute myeloid leukemia (AML), mixed-lineage leukemia (MLL) and adult T-cell
leukemia/lymphoma cells (ATL) [13–16]. These findings suggest that CDK9 could be a potential target
for cancer therapy.
Recently, several selective CDK9 inhibitors with potent in vitro and in vivo antitumor activity
have been reported [17]. 12u, LY2857785, and LDC000067 with CDK9 IC50 values of 14 nM, 11 nM,
and 44 nM, respectively, are in preclinical studies [18–21]. BAY-1143572, the first selective CDK9
inhibitor that entered clinical research in 2016, showed a CDK9 IC50 value of 13 nM which is 100-fold
more potent than that against CDK2 (IC50 = 1300 nM) [22].
Wogonin (Fig. 1), one of the bioactive flavones isolated from the Chinese herbal Huang-Qin
(Scutellaria baicalensis Georgi, commonly known as Chinese Skullcap), has been shown to have
various pharmacological activities including neuroprotective, antioxidative, anti-inflammatory, and
especially antitumor [23–25]. Importantly, wogonin has shown virtually no toxicity for normal cells at
concentrations that are lethal to tumor cells, indicating its potential for clinical application [23,26].
Recent studies have shown that wogonin can inhibit CDK9 and lead to rapid downregulation of MCL-1
in cancer cells [23]. Here, we report the design, synthesis and biological evaluation of a series of

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wogonin derivatives as potent and selective CDK9 inhibitors.
Fig. 1. Structures of some representative non-selective CDK inhibitors and selective CDK9 inhibitors.
2. Chemistry
The general synthesis of wogonin derivatives 9a−9j in Scheme 1 was adapted from a previously
described method in the literature [26-27]. In brief, pyrogallic acid 1 was first treated with benzyl
bromide to give intermediate 2, followed by oxidation with 65% HNO3 to get the corresponding
quinone 3. Then, 3 was reduced by sodium thiosulfate to obtain the phenol 4. Subsequently,
methylation of 4 was performed to obtain intermediate 5, which was debenzylated to give intermediate
6. After Friedel-Crafts acylation with different cinnamoyl chloride derivatives, the corresponding
flavones 8a−8j were formed through cyclization. The final compounds 9a−9j were prepared by
selective demethylation of intermediates 8a−8j (Scheme 1).
The chemistry for the synthesis of wogonin derivatives 20a−20d and 24a−24l is outlined in
Scheme 2. Starting from phloroglucinol 10, the Hoesch reaction was first carried out to form
intermediate 11. After selective methylation and aldol condensation with m-bromobenzaldehyde and
p-fluorobenzaldehyde, intermediates 13a−13b were obtained. Subsequently cyclization and
deprotection of two methyl groups with 48% HBr produced intermediates 15a−15b. Then, sulfate
compounds 16a−16b were produced through the Elbs oxidation reaction with potassium persulfate in

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water. Subsequently, O5-benzylation and O7-benzylation of the sulfate compound 16a followed by
hydrolysis and O8-methylation generated intermediate 18, while selective O7-benzylation of the sulfate
compound 16b followed by hydrolysis and O8-alkylation generated intermediates 22a−22d.
Intermediates 19a−19d were obtained from 18 by treatment with different amines via the
Buchwald-Hartwig coupling reaction, and the desired compounds 20a−20d were formed by
debenzylation. Intermediates 23a−23l can be easily prepared from 22a−22d by treatment with different
amines through nucleophilic aromatic substitution, and the desired compounds 24a−24l were formed
by debenzylation.
Scheme 3 illustrates the synthesis of derivatives 30a−30b. The preparation of intermediate 28
started from the treatment of intermediate 21 with 27, which can be easily obtained from
pyrrolidin-3-ol 25. The target compounds 30a−30b were produced after nucleophilic aromatic
substitution and deprotection of benzyl and carboxybenzyl groups. Scheme 4 shows the synthesis of
derivatives 36a−36b. The 7- and 5-hydroxyl of intermediate 16b were first protected with benzyl group
and p-toluenesulfonyl group, respectively, followed by hydrolysis and the Mitsunobu reaction to obtain
intermediate 33. Intermediates 35a−35b were obtained from 33 by selective deprotection of the
p-toluenesulfonyl group and nucleophilic aromatic substitution. The final compounds 36a−36b were
prepared by deprotection of the benzyl group. The synthesis of compounds 39a-39b are shown in
Scheme 5, the 2-chloromethylfuran was reacted with 21 to obtain compound 37, which was further
treated with piperazine or methylpiperazine to obtain 38a-38b in good yield (85%-90%). Next, the
deprotection of benzyl generated target compounds 39a-39b (Scheme 5).
Compounds 43a-43b were synthesized as shown in Scheme 6. Briefly, compound 21 was also
used as a starting materal and was treated with 3-chloro-2,4-pentanedione under basic conditions to
produce compound 40 followed by a condensation reaction with hydrazinium hydroxide to generate the
key intermediate 41. Then, piperazine or methylpiperazine was introduced to obtain compounds
42a-42b. Ultimately, the target compounds 43a-43b were obtained by deprotection of the benzyl group
(Scheme 6). Compound 48 was synthesized in a similar way as compound 39b, except for using ethyl
formate instead of 3-chloro-2,4-pentanedione to obtain intermediate 44 (Scheme 7). Starting form
compound 41, the target compound 51 was obtained through debenzylation, methylation and
substituted with methylpiperazin (Scheme 8). As depicted in Scheme 1-8, a total of 35 derivatives of
wogonin were synthesized and characterized based on spectral properties.

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Scheme 1. Reagents and conditions: (i) PhCH2Br, K2CO3, acetone, reflux, 48 h, 87%; (ii) AcOH, 65% HNO3, 40
○C, 4 h, 45%; (iii) Na2S2O4, CH3COOC2H5/H2O, r.t., 1 h, 37%; (iv) Me2SO4, aq NaOH, C2H5OH, r.t., 3 h, 85%; (v)
H2, Pd/C, MeOH, r.t., 8 h, 90%; (vi) Cinnamoyl chloride derivatives, BF3-Et2O, CH2Cl2, reflux, 1.5 h~6 h,
35%~80%; (vii) I2, DMSO, 120 ○C, 5 h~8 h, 30%~40%; (viii) AlCl3, CH3CN, reflux, 8 h, 65%~85%.

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Scheme 2. Reagents and conditions: (i) CH3CN, ZnCl2, HCl (g), Et2O, 0 ºC, 40 h, 71%; (ii) Me2SO4, K2CO3,
acetone, reflux, 4 h, 83%; (iii) p-fluorobenzaldehyde or m-bromobenzaldehyde, aq KOH, C2H5OH, 40 ºC, 10 h~12
h, 85%~90%; (iv) I2, DMSO, 130 ºC, 5 h~8 h, 95%~97%; (v) HBr, reflux, 60 h~72 h, 82%; (vi) K2S2O8, Me4NOH,
H2O, 30 ºC, 3 h~4 h, 60%; (vii) PhCH2Br, Cs2CO3, DMF, 90 ºC, 6 h, ; (viii) 6M HCl, r.t., 10 h, 29%~39%; (ix)
Me2SO4, K2CO3, acetone, reflux, 3 h, 90%; (x) appropriate amine, Pd2(dba)3, BINAP, t-BuOK, toluene, reflux, 5 h,
40%~70%; (xi) H2, Pd/C, THF/MeOH, 30 ºC~45 ºC, 6 h~12 h, 30%~70%; (xii) PhCH2Br, K2CO3, DMF, 50 ºC, 5
h; (xiii) Me2SO4, appropriate bromoalkane or cyclohexyl methanesulfonate, K2CO3 or Cs2CO3, DMF, 55 ºC~85 ºC,
4 h~9 h, 50%~90%; (xiv) appropriate amine, DIPEA, DMSO, 80 ºC~120 ºC, 8 h~20 h, 44%~80%.
Scheme 3. Reagents and conditions: (i) Cbz-Cl, K2CO3, CH2Cl2/H2O, r.t., 5 h, 80%; (ii) MeSO2Cl, TEA, CH2Cl2,
0 ºC, 4 h, 80%; (iii) K2CO3, DMF, 80 ºC, 8 h, 50%; (iv) appropriate amine, DIEA, DMSO, 80 ºC~120 ºC, 8 h~20 h,
44%~80%; (ii) H2, Pd/C, THF/MeOH, 30 ºC~45 ºC, 6 h~12 h, 30%~70%.
Scheme 4. Reagents and conditions: (i) (a) PhCH2Br, K2CO3, DMF, 50 ºC, 5 h, (b) TsCl, DMAP, Cs2CO3, DMF,
50 ºC, 4 h; (ii) 6M HCl, r.t., 10 h, 20%; (iii) 1-methylpiperidin-4-ol, DEAD, PPh3, THF, r.t., 1 h, 75%; (iv) KOH,
MeOH, reflux, 90%; (v) appropriate amine, DIEA, DMSO, 80 ºC~120 ºC, 8 h~20 h, 44%~80%; (ii) H2, Pd/C,
THF/MeOH, 30 ºC~45 ºC, 6 h~12 h, 30%~70%.

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Scheme 5. Reagents and conditions: (i) 2-(chloromethyl)furan, K2CO3, CH3CN, reflux, 2 h, 60%; (ii) appropriate
amine, DIEA, DMSO, 80 ºC~120 ºC, 8 h~20 h, 44%~80%; (iii) H2, Pd/C, THF/MeOH, 15 ºC, 6 h~12 h,
30%~70%.
Scheme 6. Reagents and conditions: (i) 3-chloro-2,4-pentanedione, K2CO3, acetone, reflux, 3 h, 50%; (ii)
hydrazinium hydroxide, EtOH, CH3COOH, r.t., 90%; (iii) appropriate amine, DIEA, DMSO, 80 ºC~120 ºC, 8
h~20 h, 44%~80%; (iv) H2, Pd/C, THF/MeOH, 30 ºC~45 ºC, 6 h~12 h, 30%~70%.
Scheme 7. Reagents and conditions: (i) Ethyl chloroacetate, K2CO3, acetone, reflux, 4 h, 60%; (ii) Ethyl formate,
NaH, THF, 45 ºC, 4 h, 55%; (iii) hydrazinium hydroxide, MeOH, r.t., 70%; (iv) 1-methylpiperazine, DIEA, DMSO,
85 ºC, 8 h, 45%; (v) H2, Pd/C, THF/MeOH, 30 ºC, 8 h, 40%.
Scheme 8. Reagents and conditions: (i) H2, Pd/C, THF/MeOH, 30 ºC, 6 h; (ii) Me2SO4, K2CO3, acetone, 50 ºC, 3 h,

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65%. (iii) methylpiperazine, DIEA, DMSO, 60 to 80 ºC, 8 h, 68.5%.
3. Results and discussion
3.1. Rational drug design
Due to the evolutionary conservation of the ATP binding site, the development of highly selective
small molecule ATP-antagonistic CDK inhibitors is not an easy task [20, 28]. CDK9 specificity is not
easy to achieve due to its cross-reaction with other cell cycle CDKs, in particular CDK2. By analyzing
the crystal structures of CDK2 and CDK9, we found that the ATP binding pocket of CDK9 is more
flexible than that of CDK2, indicating that the CDK9 ATP pocket can accommodate larger ligands (Fig.
2). Therefore, in this study, a series of derivatives with substituents at the para or meta position of the
B ring of wogonin were designed and synthesized to obtain selective CDK9 inhibitors.
Fig. 2. (A) The comparison of the ATP binding pocket in CDK9 and CDK2. (B) The binding mode of
wogonin in CDK9 and CDK2.
3.2. Structure-activity relationship analysis
We first designed and synthesized several derivatives with different electron withdrawing or
donating groups on the B ring of wogonin to explore the effect on its potency and selectivity for CDK9.
CDK2 was used as a control since it has a high sequence identity with other CDKs. All compounds
were tested against CDK9 and CDK2 (at a concentration of 1 µM), as well as the hepatoma cells cell
line HepG2. The results are summarized in Table 1, together with the potencies of the inhibitory effects
of wogonin as reference. It was found that introduction of electron withdrawing or donating
substituents at different positions of the B ring of wogonin offer neither obvious enhancement in the
kinase activity nor in the anticancer activity. In particular, electron donating groups, such as methyl or
methoxyl (compound 9c and 9d), have detrimental effects on the inhibitory activity.

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Table 1
In vitro activity of wogonin derivatives with B-ring modification
Kinase inhibition (%)a
Cytotoxicity
Comp.
R
IC50/µMb, HepG2
CDK9/T1
67.8
CDK2/A
40.7
ND
-
17.6
34.2
>100
24.9
42.4
59.4
38.2
>100
>100
33.5
wogonin
9a
p-Cl
46.4
p-NO2
p-CH3
p-OCH3
m-F
32.5
ND
9b
19.2
ND
9c
27.6
ND
9d
56.2
ND
9e
m-Br
m-CF3
o-Cl
42.6
ND
9f
52.3
ND
9g
51.4
ND
9h
o-Br
56.5
18.9
9i
o-CF3
54.6
ND
>100
9j
a Percent inhibition at 1 µM, values are the averages derived from at least three replicates.
b Anti-proliferative activity by MTT-72 h assay, values are the averages from at least five independent dose
response curves.
Then, a series of derivatives with bulkier substituents at the para or meta position of the B ring of
wogonin were additionally designed and synthesized. According to the data presented in Table 2,
compounds 20a−20d and 24a−24f exhibited enhanced selectivity for CDK9 compared to wogonin.
Generally, the CDK9 inhibitory activity of the para-substituted compounds 24a−24f was significantly
higher than that of the meta-substituted compounds 20a−20d. In addition, among these compounds, the
piperazine and methylpiperazine substituted analogues 20b, 20c, 24d and 24e exhibited potent
proliferstion inhibitory activity in the HepG2 cell line, while the morpholine and pyrrolidine substituted
analogues 20a, 24a and 24c showed dramatically reduced antiproliferative activity. Compound 24e was
the most potent and selective CDK9 inhibitor in this series with a HepG2 IC50 value of 6.6 µM.
Table 2

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In vitro activity of wogonin derivatives with further B-ring modification
Kinase inhibition (%)a
Cytotoxicity
IC50/µMb, HepG2
Comp.
R
CDK9/T1
45.4
CDK2/A
NA
m-morpholino
m-piperazin-1-yl
m-methylpiperazin-1-yl
m-1,4-diazepan-1-yl
p-pyrrolidin-1-yl
p-diethylamino
>100
9.9
20a
20b
20c
20d
24a
24b
24c
24d
24e
46.4
NA
46.5
12.2
11.9
NA
13.1
21.7
>100
6.4
60.9
33.9
58.0
29.2
NA
p-morpholino
58.0
78.3
17.9
6.6
p-piperazin-1-yl
72.1
NA
p-methylpiperazin-1-yl
81.2
NA
p-1,4-diazepan-1-yl
65.4
12.7
18.2
24f
a Percent inhibition at 1µM, values are the averages derived from at least three replicates.
b Anti-proliferative activity by MTT-72 h assay, values are the averages from at least five independent dose
response curves.
In order to improve the kinase inhibitory activity and cellular antiproliferative potency, we further
modified the 8-methoxy group of wogonin based on activity data collected from previous analogues.
Different polar and nonpolar groups were introduced to the O8 of wogonin, while piperazine and
methylpiperazine were introduced to the para-position of the B ring. In vitro enzyme activity assay
showed that this series of compounds had a similar CDK9 selectivity profile, but their inhibitory
activity varied widely (Table 3). The O8-isopropyl substituted analogues were more potent than the
corresponding O8-cyclopentyl, O8-cyclohexyl and furylmethyl substituted analogues, as indicated by
compounds 24g, 24i, 24l and 42a. This finding suggests that it is difficult to accommodate large-sized
hydrophobic groups in the cavity where the wogonin O8 is located. Modification of hydrophilic groups
like pyrrolidinyl, methylpiperidinyl, and dimethylpyrazolyl at the O8-position resulted in compounds
30a−30b, 36a−36b and 43a-43b, which showed comparable selectivity and inhibitory activity for
CDK9, but their cellular potency increased dramatically. Further determination of the CDK9 and
CDK2 IC50 values of the representative compounds 24g, 24h, 43a and 43b showed that their CDK9
inhibitory activities were significantly improved compared to wogonin (Table 4). In particular,

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compound 43a was the most potent CDK9 inhibitor with an IC50 value of 10.8 nM, which was ten-fold
higher than that of wogonin. On the other hand, they had weak inhibitory activity against CDK2.
Table 3
In vitro activity of wogonin derivatives with 8-O-modification
Kinase inhibition (%)a
cytotoxicity
Comp.
24g
24h
24i
R1
R2
IC50/µMb, HepG2
CDK9/T1
CDK2/A
p-piperazin-1-yl
88.6
31.4
18.0
p-methylpiperazin-1-yl
p-piperazin-1-yl
82.1
14.1
30.7
52.1
56.5
74.0
57.0
24.0
NDc
ND
ND
ND
NAd
NA
14.8
5.3
p-methylpiperazin-1-yl
p-piperazin-1-yl
15.8
22.8
27.8
>100
>100
24j
24k
24l
p-methylpiperazin-1-yl
p-piperazin-1-yl
30a
30b
p-methylpiperazin-1-yl
p--piperazin-1-yl
p-methylpiperazin-1-yl
p-piperazin-1-yl
74.6
71.8
40.2
52.6
97.1
NA
NA
NA
NA
32.2
39.4
6.7
8.3
7.0
8.5
36a
36b
39a
39b
43a
p-methylpiperazin-1-yl
p-piperazin-1-yl

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p-methylpiperazin-1-yl
p-methylpiperazin-1-yl
98.1
51.1
ND
6.3
8.1
43b
48
34.0
a Percent inhibition at 1 µM, values are the averages derived from at least three replicates.
b Anti-proliferative activity by MTT-72 h assay, values are the averages from at least five independent dose
response curves.
c ND represents not determine.
d NA represents no activity.
Table 4
Inhibition (IC50) of representative compounds 24g-24h, 43a-43b and wogonin against CDK9, CDK2
and cancer cells HepG2, A549, HCT116 and MV4-11.
Kinase inhibition IC50
(nM)a
Comp.
Structure
HepG2
A549
HCT116
MV4-11
CDK9/T1 CDK2/A
198
94.1
179
1460
NA
17.6
42.3
27.3
30.7
wogonin
17.9
17.7
8.6
5.5
4.1
5.5
2.5
3.4
12.8
4.8
6.1
6.3
4.4
2.2
24g
24h
43a
43b
NA
10.8
12.7
1040
830
6.3
3.3
a Values are the averages from three-independent experiments.

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3.3. Cellular antiproliferative activity towards a panel of cancer cell lines
The cellular antiproliferative activity of representative compounds was further evaluated in a
panel of human cancer cell lines including MV4-11 (leukemia), A549 (lung), HCT116 (colon) and
MCF-7 (breast) using the tetrazolium dye (MTT; 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) assay (Table 4). Wogonin was selected as the control and the inhibitory activity of wogonin
toward these cancer cells was similar to that reported by others [29-31]. Most of the representative
compounds showed potent inhibitory activity toward all four cell lines. Especially compounds 43a and
43b, which were active against all four cell lines, and showed much improved inhibitory activity
toward these cell lines compared with wogonin. It is worth noting that compound 43a and 43b
exhibited the strongest inhibitory effect against CDK9 with an IC50 value of 10.8 and 12.7 nM,
respectively.
3.4. Drug-like properties-inspired optimization
By combining the selectivity for CDK9 and inhibitory activity against cancer cells, we intended to
select compound 43a for further in vivo evaluation. However, this compound showed poor
physicochemical properties, especially low solubility in water (1.0 µg/mL), which may limit its
application in further in vivo studies. Therefore, for further in vivo evaluation studies, it was necessary
to improve the solubility of this compound. By analyzing its structure, it was suspected that the
presence of the two hydroxyls may limit its drug-like property. The hydroxyl at the 5-position formed
an important hydrogen bond with CDK9, while the C-7 hydroxyl did not show a crucial role in the
binding with CDK9. Thus, a methyl group was introduced at the C-7 hydroxyl position to obtain
compound 51. The solubility and Log D7.4 were determined for compounds 43a and 51. Wogonin was
selected as the control. Two methods were used to determine the solubility. Their intrinsic aqueous
solubility was determined on a Gemini Profiler instrument (pION Inc., Woburn, MA, USA) using the
‘goldstandard’ Avdeef-Bucher potentiometric titration method. Compound 51 (28 µg/mL) showed a
28-fold higher solubility than compound 43a without methyl at C-7. Another method was used to
determine their solubility under acidic condition, compound 51 showed dramatically increased
solubility (solubility was determined in diluted methanesulfonic acid buffer at pH = 4.5). The
hydrophobicity parameter Log D7.4 of compounds 51 was also found to be more acceptable than that of
compound 43a. (Table 5). In addition, compound 51 showed comparable inhibitory activity against
CDK9 and selectivity towards CDK2. Thus, compound 51, which showed improved physicochemical
properties and potent CDK9 inhibitory activity, was selected for further pharmacological evaluation
both in vitro and in vivo.

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Table 5
Physicochemical properties and inhibition against CDKs of compounds 43a, 51 and Wogonin.
Intrinsic
solubility
Solubility
(pH = 4.5)
（µg/mL）
Log D
CDK9
CDK2
Comp.
Structure
(pH = 7.4)
IC50 (nM)
IC50 (nM)
（µg/mL）
43a
1.0
30
2.91
10.8
1040
51
28
13
4540
149
2.01
1.18
19.9
198
913
-
Wogonin
1460
3.5. Molecular docking
The molecular docking studies were performed to understand the interaction pattern of the
representative compound 51 in the active site of CDK9 (PDB ID: 3BLR). The overlay of 51 with
flavopiridol in the binding site is shown in Fig. 3A. The key interactions of 51 with the key residues in
the active site are shown in Fig. 3B and 3C. As depicted in Fig. 3B, compound 51 could well embed in
the ATP binding pocket of CDK9. The carbonyl group of compound 51 forms a hydrogen bond with
Cys106 in the hinge region. In addition, compound 51 forms another important H-bonds with Ala153.
Besides these H-bond interactions, two other important H-π interactions, specifically with Phe103 and
Leu156, were also observed. The benzomethylpiperazine moiety could easily reach the solvent area.
When docking the compound into the CDK2 ATP binding site (PDB ID: 4BCP, Fig. 3D), the steric
hindrance of Lys89 at the inlet of the ATP binding pocket makes it difficult for compound 51 to form a
stable conformation and thus cannot bind tightly to CDK2. The corresponding amino acid in the CDK9
active site is Gly112, which is more favorable for accommodating the piperazinyl of compound 51.
This difference in binding conformations may be the reason for the high potency and selectivity of
compound 51 toward CDK9.

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Fig. 3. (A) Overlay of flavopiridol (yellow) with compound 51 (brown) in the active site of CDK9. (B)
The docking model of compound 51 in a complex with CDK9. The hydrogen bonds ares depicted by
black dotted lines. (C) Two-dimensional view of the key interactions of 51 with the active site residues.
(D) The active site of CDK2. The steric hindrance of Lys89 is shown as surface.
.
Table 7
Antiproliferative activity of compound 51 against a panel of human tumor cell lines
Human cell line
Human cell line
cytotoxicity
cytotoxicity
IC50 (µM)a
IC50 (µM)a
Origin
Designation
HL60
Origin
Designation
HT29
Leukemia
Leukemia
5.21
0.02
Colon carcinoma
Colon carcinoma
1.80
1.86
MV4-11
SW480
Leukemia
Leukemia
CEM
1.24
0.79
Lung carcinoma
A549
1.81
3.12
THP-1
Gastric carcinoma
BGC803
Cervical
Breast carcinoma
MCF-7
0.48
Hela
2.32
carcinoma
Breast carcinoma
Hepatic carcinoma
BT-549
HepG2
1.85
2.67
Myeloma
RPMI8226
HEK293
0.82
22.1
Renal (normal)

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Colon carcinoma
HCT-116
0.39
Liver (normal)
L02
15.1
a
Anti-proliferative activity by MTT-72 h assay, values are the averages from at least five independent dose
response curves.
3.6. Evaluation of the cellular inhibitory effect and mechanism of action of 51
The cellular antiproliferative activity of compound 51 was analyzed in a panel of 14 cancer cell
lines (CDK9-expressing) and 2 normal cell lines (Table 7). The analysis revealed that compound 51
had potent cellular antiproliferative activity against these cancer cell lines, especially against the
leukemia cancer cell line MV4-11, with an IC50 value of 20 nM. When compound 51 was used to treat
the normal cell lines (HEK293 and L02), it exhibited low toxicity (IC50 values of 15 and 22 µM). It is
worth noting that the introduction of methyl in the 7-position of compound 51, it significantly
improved the antiproliferative activity against certain types of tumor cell lines, including MV4-11 and
HCT116, compared with 43a. The evaluation of the inhibition of cellular showed that compound 51 has
a good cellular therapeutic window, with a superior profile to that of flavopiridol. Compared with
flavonoid derivatives previously reported by our collaborative group, compound 51 exhibited the best
in vitro antitumor activity profile [32-33].
Based on the results of the analysis of the in vitro antitumor activity, the MV4-11 cell line was
selected to examine the effects of compound 51 on the CDK9-mediated signaling pathways (Fig. 4).
The effect of the treatment with compound 51 on the distribution of cells in the cell cycle was first
assessed by propidium iodide (PI) staining. Synchronized cells were r left untreated as control or
treated with compound 51. Compound 51 (at 0.25 or 1.25 µM) induced an obvious increase in the
number of cells in the G1 phase with a decrease was detected in the S phase at 24 h, which may be due
to the higher selectivity of compound 51 toward the inhibition of CDK9 (a transcriptional regulator, not
a cell cycle regulator).

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Fig. 4. Effects of compound 51 on cell cycle progression. (A) the control. (B) 51 (0.05 µM). (C) 51
(0.25 µM). (D) 51 (1.25 µM).
We also evaluated apoptosis in MV4-11 cell line after treatment with the indicated concentrations
of 51 for 24 h. The nuclei changes in MV4-11 cells were observed by confocal microscopy, after
staining with DAPI. As shown in Fig. 5A, untreated MV4-11 cells were stained uniformly with blue
fluorescence, while compound 51-treated cells emitted bright fluorescence, which indicates the early
phenomena of apoptosis. In addition, the nuclei showed a dose-dependent and time-dependent
chromatin condensation and characteristic apoptotic morphological changes when treated with 51 (Fig.
5B). To further confirm the compound 51-induced apoptosis, the Annexin V/PI staining assay was
employed. After cells were treated with 0.05, 0.25 and 1.25 µM of compound 51 for 24 h, the
percentage of total apoptotic cells was 17.95, 28.54 and 35.48%, respectively, indicating the induction
of apoptosis in a concentration-dependent manner (Fig. 5C). Together, these results demonstrated that
compound 51 inhibited proliferation of MV4-11 cells through a concentration- and time-dependent
apoptotic mechanism.

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Fig. 5. Effect of compound 51 on apoptosis in MV4-11 cells by microscopy with DAPI staining after
treatment with compound 51 for 24 h (A) or 48 h (B), and by flow cytometry (C).
Western blot analysis was performed to study the cell proliferation inhibitory mechanism. As
expected, upon 4 h treatment with compound 51, the direct phosphorylation site in the
carboxy-terminal domain of phosphorylated-RNA Pol II Ser2 was dose-dependently inhibited. In
addition, the expression level of proteins, such MCL-1 and c-MYC were dose-dependently decreased.
MCL-1 and c-MYC have been reported to be important antiapoptotic proteins, which could hinder
apoptosis in cancer cells (Fig. 6). In contrast, the antiapoptotic protein BCL-2 was not affected when
treated with compound 51. Furthermore, we also analyzed the effects of compound 51 on important
apoptosis markers. After treatment for 24 h with compound 51, a dose-dependent increase in the
protein levels of apoptosis proteins cleaved PARP and caspase-3 was observed (Fig. 6). Together, all
the results suggested that in MV4-11 cells compound 51 can induce apoptosis by targeting CDK9.
Fig. 6. Cellular effects of compound 51 on CDK9-mediated signaling pathways and apoptosis in
MV4-11 cells.
3.7. In vivo evaluation of compound 51.
We also evaluated the in vivo antitumor activity of compound 51 in the subcutaneous MV4-11
AML xenograft murine model. The nude mice bearing MV4-11 tumor xenografts were randomly

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injected with vehicle, the positive control drug doxorubicin (5 mg/kg) and compound 51 (15, 30 and 45
mg/kg) every other day for three weels. The treatment with compound 51 resulted in the marked
inhibition of tumor growth (Fig. 7A, B). On day 21 of treatment with compound 51 (15, 30 and 45
mg/kg), the tumor growth inhibition (TGI) was 22.9, 42.6 and 53.0%, respectively, without cansing any
mortality. In addition, there was no significant change in the body weight after treatment with 51 at
various concentrations. Thus, the in vivo study results further confirmed the antitumor potential of
compound 51.
Fig. 7. In vivo anticancer efficacy of compound 51. (A) compound 51 and the positive control
doxorubicin suppressed the tumor growth in vivo in MV4-11 tumor xenografts nude model. (B) Tumor
volumes of each mouse on day 21.
4. Conclusion
In this study, inspired by the selective CDK9 flavonoid antitumor compound Wogonin, a series of
derivatives were designed and synthesized. Among them, a promising compound, namely compound
51 (named as LBJ-23) emerged as the most specific inhibitor of CDK9 with an IC50 value of 19.9 nM
and exhibited excellent selectivity toward other CDKs. The preliminary docking studies may explain
the selectivity toward CDK9. Additionally, it also shows an acceptable drug-like properties with good
solubility both in aqueous and weak acid environment. Compound 51 exhibited strong antiproliferative
activity against a panel of established cell lines including leukemia cell lines and solid tumor cell lines
by inducing apoptotic cell death. Furthermore, the highlighted compound 51 showed significant
antitumor efficacy without obvious toxic effects in vivo, which provide us a new chemical tool for
further investigate research CDK9-targeted inhibitors as a potential antitumor drug, especially for
AML.

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5. Experimental procedures
5.1. Chemistry
General experimental methods. All chemicals and reagents and were purchased from commercial
o
sources. Organic solutions were concentrated in a rotary evaporator (Büchi Rotavapor) below 55 C
under reduced pressure. Silica gel thin-layer chromatography was performed on precoated plates
GF-254 and visualized under UV light. Melting points were determined with a Melt-Temp II apparatus.
1H NMR and 13C NMR spectra were recorded on a Bruker AV-300 instrument using deuterated solvents
with tetramethylsilane (TMS) as internal standard. Chemical shifts are given in ppm (δ). The
multiplicities are denoted as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet; br s, broad
singlet. IR spectra were recorded on a Nicolet iS10 Avatar FT-IR spectrometer using KBr film.
ESI-mass and high resolution mass spectra (HRMS) were recorded on a Water Q-Tofmicro mass
spectrometer. All tested compounds exhibited greater than 95% purity unless otherwise noted. The
synthesis routes of 9a-9j were shown in previos works [26-27].
5.1.1. 2, 4, 6-Trihydroxyacetophenone hydrate (11).
Pyrogallol (10) (63 g, 0.5 mol) was dissolved in anhydrous ether (300 mL), then anhydrous ZnCl2
(13.4 g, 0.1 mol) and anhydrous acetonitrile (50 g, 1.25 mol) were added and then the reaction mixture
o
was stirred in the presence of dried HCl gas at 0 C for 40 h. The reaction was monitored by TLC. After
the end of the reaction, the mixture was raised to room temperature and filtered. The filter cake was
dried to constant weight and dissolved in water (200 mL). Then the mixture was refluxed for 6 h and a
large amount of solid was formed. The mixture was cooled to room temperature, filtered, and the cake
was dried to give a white solid (60 g, 71% yield). mp 119-121 oC.
5.1.2. 4, 6-Dimethoxy-2-hydroxyacetophenone (12).
Compound 11 (33.6 g, 0.2 mol) was dissolved in anhydrous acetone (350 mL) and K2CO3 (63.48
g, 0.46 mol) was added. Then dimethyl sulphate was added dropwise at room temperature and the
mixture was heated to 45 oC for 4 h. The reaction was monitored by TLC. After the end of the reaction,
the mixture was cooled to room temperature, filtered and the filtrate was poured into 5 volumes of
water and adjusted to pH = 3 - 4 with 1 M HCl to precipitate a large amount of solid. The mixture was
filtered and dried to give a white solid (32.5 g, 83% yield). mp 80-82 oC.
5.1.3. (E)-3-(3-Bromophenyl)-1-(2-hydroxy-4, 6-dimethoxyphenyl)prop-2-en-1-one (13a).
Compound 12 (30 g, 0.15 mol) was dissolved in ethanol (450 mL) and p-fluorobenzaldehyde (31
g, 0.17 mol) was added. Then 7% KOH aqueous solution (280 mL, 0.38 mol) was added dropwise and
the mixture was kept below 5 oC and then heated to 40 oC for 12 h. The reaction was monitored by TLC.
After the end of the reaction, the mixture was cooled to room temperature and poured into 10 volumes

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of water and adjusted to pH = 3 - 4 with 6 M HCl to precipitate a large amount of solid. The mixture
was filtered and dried to give a yellow solid (50.1 g, 90% yield). mp 190-193 oC.
5.1.4. (E)-3-(4-Fluorophenyl)-1-(2-hydroxy-4, 6-dimethoxyphenyl)prop-2-en-1-one (13b).
Compound 12 (16.36 g, 84 mmol) was dissolved in ethanol (250 mL) and p-fluorobenzaldehyde
(11.39 g, 92 mmol) was added. Then 7% KOH aqueous solution (150 mL, 0.21 mol) was added
dropwise and the mixture was kept below 5 oC and then heated to 40 oC for 12 h. The reaction was
monitored by TLC. After the end of the reaction, the mixture was cooled to room temperature and
poured into 10 volumes of water and adjusted to pH = 3-4 with 6 M HCl to precipitate a large amount
of solid. The mixture was filtered and dried to give a yellow solid (21.33 g, 84.6% yield). mp 200-202
oC.
5.1.5. 2-(3-Bromophenyl)-5, 7-dimethoxy-4H-chromen-4-one (14a).
Compound 13a (16.7 g, 46 mmol) was dissolved in DMSO (40 mL) at the temperature of 60 oC
and I2 (cat., 0.32 g, 1.2 mmol) was added. Then the mixture was heated to 130 oC for 8 h. The reaction
was monitored by TLC. After the end of the reaction, the hot mixture was poured into 10 volumes of a
saturated aqueous solution of sodium thiosulfate to precipitate a large amount of solid. The mixture was
filtered and dried to give a yellow solid (16.2 g, 97% yield). mp 278-280 oC.
5.1.6. 2-(4-Fluorophenyl)-5, 7-dimethoxy-4H-chromen-4-one (14b).
Compound 13b (17.33 g, 57 mmol) was dissolved in DMSO (43 mL) at the temperature of 60 oC
and I2 (cat., 0.36 g, 1.4 mmol) was added. Then the mixture was heated to 130 oC for 5 h. The reaction
was monitored by TLC. After the end of the reaction, the hot mixture was poured into 10 volumes of a
saturated aqueous solution of sodium thiosulfate to precipitate a large amount of solid. The mixture was
filtered and dried to give a yellow solid (16.3 g, 95% yield). mp 291-295 oC.
5.1.7. 2-(3-Bromophenyl)-5, 7-dihydroxy-4H-chromen-4-one (15a).
Compound 14a (20 g, 55 mmol) and 48% hydrobromide (250 mL) were refluxed for 72 h and the
reaction was monitored by TLC. After the end of the reaction, the mixture was cooled to room
temperature and poured into 10 volumes of ice water to precipitate a large amount of solid. The mixture
was filtered and the cake was washed with water until the pH was neutral, dried to give a yellow solid
(15.1 g, 82% yield). mp 192-196 oC.
5.1.8. 2-(4-Fluorophenyl)-5, 7-dihydroxy-4H-chromen-4-one (15b).
Compound 14b (21.36 g, 71 mmol) and 48% hydrobromide (300 mL) were refluxed for 60 h and
the reaction was monitored by TLC. After the end of the reaction, the mixture was cooled to room
temperature and poured into 10 volumes of ice water to precipitate a large amount of solid. The mixture
was filtered and the cake was washed with water until the pH was neutral, dried to give a crude product
(20.9 g). The solid was purified by polyamide column chromatography to give a yellow solid (16.2 g,

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83% yield). mp 206-210 oC.
5.1.9. Tetramethylammonium-5, 7-dihydroxy-4-oxo-2-(3-bromophenyl)-4H-chromen-8-yl sulfate (16a).
Compound 15a (21.2 g, 63.7 mmol) and tetramethylammonium hydroxide (69.19 g, 38.2 mmol)
were dissolved in water (530 mL) and potassium persulfate (42.93 g, 169 mmol) was added eight times
every 20 minutes. The mixture was kept below 30 oC for 3.5 h and the reaction was monitored by TLC.
After the end of the reaction, the mixture was filtered and the filtrate was adjusted to pH = 6 - 7 with
potassium dihydrogen phosphate (about 30 g) to precipitate solid. Then sodium chloride (45 g) was
added three times every 5 minutes and the mixture was stirred at room temperature overnight. The
mixture was filtered and the cake was washed with methanol (20 mL) and dried to give a reddish
brown solid (19.2 g, 60.1% yield).
5.1.10. Tetramethylammonium-5, 7-dihydroxy-4-oxo-2-(4-fluorophenyl)-4H-chromen-8-yl sulfate (16b).
Compound 15b (18 g, 71 mmol) and tetramethylammonium hydroxide (71.89 g, 40 mmol) were
dissolved in water (450 mL) and potassium persulfate (35.7 g, 130 mmol) was added eight times every
20 minutes. The mixture was kept below 30 oC for 3 h and the reaction was monitored by TLC. After
the end of the reaction, the mixture was filtered and the filtrate was adjusted to pH = 6 - 7 with
potassium dihydrogen phosphate (about 30 g) to precipitate solid. Then sodium chloride (45 g) was
added three times every 5 minutes and the mixture was stirred at room temperature overnight. The
mixture was filtered and the cake was washed with methanol (20 mL) and dried to give a yellowish
solid (17 g, 60.8% yield).
5.1.11. 5, 7-Bis(benzyloxy)-2-(3-bromophenyl)-8-hydroxy-4H-chromen-4-one (17).
Compound 16a (5 g, 10 mmol) was dissolved in DMF (35 mL) and Cs2CO3 (19.4 g, 60 mmol)
was added. Benzyl bromide (7 mL, 60 mmol) was added dropwise and then the mixture was heated to
90 oC for 6 h. The reaction was monitored by TLC. After the end of the reaction, the mixture was
cooled to room temperature, filtered, and the cake was washed with methanol (20 mL). The filtrate was
adjusted to pH = 1 - 2 with 6 M HCl and the mixture was stirred at room temperature overnight to
precipitate a large amount of solid. Then the mixture was filtered and the cake was washed with
methanol (10 mL) and dried to give a yellow solid (1.5 g, 28.5% yield). mp 290-292 oC. 1H NMR (300
MHz, DMSO-d6):
δ = 9.10 (s, 1H, 8-OH), 8.35 (s, 1H, Ar-H), 7.90 (m, 3H, Ar-H), 7.50 (m, 10H, Ar-H),
6.94 (s, 1H, CHCO), 6.86 (s, 1H, Ar-H), 5.34 (s, 2H, OCH2-Ar), 5.17 (s, 2H, OCH2-Ar) ppm.
5.1.12. 5, 7-Bis(benzyloxy)-2-(3-bromophenyl)-8-methoxy-4H-chromen-4-one (18).
Compound 17 (200 mg, 0.38 mmol) was dissolved in acetone (40 mL) and K2CO3 (104.3 mg, 0.76
mmol) and dimethyl sulfate (71.5 mg, 0.57 mmol) were added. The mixture was refluxed for 3 h and
the reaction was monitored by TLC. After the end of the reaction, the mixture was cooled to room
temperature, filtered, and the cake was washed with dichloromethane (10 mL). The filtrate was

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evaporated under reduced pressure. The crude products were recrystallized from mixed solvent of
hexane and dichloromethane to give a yellow solid (185 mg, 90% yield). mp 230-233 oC. 1H NMR (300
MHz, DMSO-d6):
δ = 8.12 (s, 1H, Ar-H), 7.42 (m, 13H, Ar-H), 6.95 (s, 1H, CHCO), 6.83 (s, 1H, Ar-H),
5.32 (s, 2H, OCH2-Ar), 5.20 (s, 2H, OCH2-Ar), 3.85 (s, 3H, ArOCH3) ppm.
5.1.13. 5, 7-Bis(benzyloxy)-8-methoxy-2-(3-morpholinophenyl)-4H-chromen-4-one (19a).
Pd2(dba)3 (21.1 mg, 0.023 mmol) and BINAP (28.65 mg, 0.046 mmol) were added in anhydrous
toluene (30 mL). The mixture was stirred at room temperature for 15 minutes under N2 protection, then
compound 18 (250 mg, 0.46 mmol), morpholine (80.16 mg, 0.92 mmol) and potassium t-butoxide
(103.25 mg, 0.92 mmol) were added. The mixture was refluxed for 5 h and the reaction was monitored
by TLC. After the end of the reaction, the mixture was cooled to room temperature and evaporated
under reduced pressure. The crude products were purified over silica gel column chromatography (#
o
1
100-200) to give a yellow solid (185 mg, 73.2% yield). mp 230-233 C. H NMR (300 MHz,
DMSO-d6): = 8.21 (s, 1H, Ar-H), 7.53 (m, 13H, Ar-H), 7.01 (s, 1H, CHCO), 6.89 (s, 1H, Ar-H), 5.32
δ
(s, 2H, OCH2-Ar), 5.24 (s, 2H, OCH2-Ar), 3.83 (s, 3H, ArOCH3), 3.74 (s, 4H, N(CH2CH2)2O), 3.28 (s,
4H, N(CH2CH2)2O) ppm.
5.1.14. 5, 7-Bis(benzyloxy)-8-methoxy-2-(3-(piperazin-1-yl)phenyl)-4H-chromen-4-one (19b).
o
Yellow solid (100 mg, 57% yield). mp 225~227 C. 1H NMR (300 MHz, DMSO-d6):
δ = 8.21 (s,
1H, Ar-H), 7.55 (m, 13H, Ar-H), 6.98 (s, 1H, CHCO), 6.87 (s, 1H, Ar-H), 5.32 (s, 2H, OCH2-Ar), 5.25
(s, 2H, OCH2-Ar), 3.83 (s, 3H, ArOCH3), 3.20 (s, 4H, N(CH2CH2)2NH), 2.91 (s, 4H, N(CH2CH2)2NH)
ppm.
5.1.15. 5, 7-Bis(benzyloxy)-8-methoxy-2-(3-(4-methylpiperazin-1-yl)phenyl)-4H-chromen-4-one (19c).
o
Yellow solid (135 mg, 65% yield). mp 230~233 C. 1H NMR (300 MHz, DMSO-d6):
δ = 8.21 (s,
1H, Ar-H), 7.60 (m, 13H, Ar-H), 7.00 (s, 1H, CHCO), 6.89 (s, 1H, Ar-H), 5.32 (s, 2H, OCH2-Ar), 5.26
(s, 2H, OCH2-Ar), 3.84 (s, 3H, ArOCH3), 3.36 (s, 4H, Ar-N(CH2CH2)2N), 3.00 (s, 3H, NCH3), 2.75 (s,
4H, Ar-N(CH2CH2)2N) ppm.
5.1.16. 5, 7-Bis(benzyloxy)-2-(3-(1, 4-diazepan-1-yl)phenyl)-8-methoxy-4H-chromen-4-one (19d).
Yellow solid (110 mg, 37% yield). mp 235~237 oC. 1H NMR (300 MHz, DMSO-d6):
δ = 8.10 (s,
1H, Ar-H), 7.53 (m, 13H, Ar-H), 7.00 (s, 1H, CHCO), 6.87 (s, 1H, Ar-H), 5.28 (s, 2H, OCH2-Ar), 5.19
(s, 2H, OCH2-Ar), 3.86 (s, 3H, ArOCH3), 3.62 (s, 4H, Ar-N(CH2)2), 2.95 (s, 2H, NHCH2), 2.70 (s, 2H,
NHCH2), 1.84 (m, 2H, NCH2CH2CH2NH) ppm.
5.1.17. 5, 7-Dihydroxy-8-methoxy-2-(3-morpholinophenyl)-4H-chromen-4-one (20a).
Compound 19a (185 mg, 0.27 mmol), Pd/C (60 mg) and tetrahydrofuran (10 mL) were added in
methanol (10 mL). The mixture was stirred in the presence of H2 and heated to 35 oC for 6 h. The
reaction was monitored by TLC. After the end of the reaction, the mixture was cooled to room

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temperature and filtered. The filtrate was evaporated under reduced pressure and purified over silica gel
o
column chromatography (# 100-200) to give a yellow solid (78 mg, 77.3% yield). mp 204~206 C.
1
HRMS (ESI+) calcd for C20H20NO6 [M + H]+: 370.1291, found 370.1271. H NMR (300 MHz,
DMSO-d6):
7.50 Hz, Ar-H), 7.05 (s, 1H, CHCO), 6.30 (s, 1H, Ar-H), 3.85 (s, 3H, ArOCH3), 3.77 (s, 4H,
N(CH2CH2)2O), 3.23 (s, 4H, N(CH2CH2)2O) ppm. 13C NMR (75 MHz, DMSO-d6):
= 182.1, 163.4,
δ = 12.53 (s, 1H, 5-OH), 10.81 (s, 1H, 7-OH), 7.53-7.41 (m, 3H, Ar-H), 7.20 (d, 1H, J=
δ
157.3, 156.2, 151.5, 149.6, 131.6, 129.8, 127.6, 118.7, 116.7, 112.2, 105.0, 103.7, 99.0, 66.0, 61.0 (2 ×
C), 48.0 (2 × C) ppm. IR (KBr) ν 3245, 2829, 1657, 1586, 1507, 1444, 1356, 1251, 1163, 1023, 1010,
863, 837, 699, 671 cm-1.
5.1.18. 5, 7-Dihydroxy-8-methoxy-2-(3-(piperazin-1-yl)phenyl)-4H-chromen-4-one (20b).
o
Yellow solid (30 mg, 44.7% yield). mp 230~232 C. HRMS (ESI+) calcd for C20H21N2O5 [M +
H]+: 369.1450, found 369.1439. 1H NMR (300 MHz, DMSO-d6):
Ar-H), 7.18 (d, 1H, J= 7.20 Hz, Ar-H), 7.00 (s, 1H, CHCO), 6.26 (s, 1H, Ar-H), 3.84 (s, 3H, ArOCH3),
3.20 (s, 4H, N(CH2CH2)2NH), 2.90 (s, 4H, N(CH2CH2)2NH) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 12.51 (s, 1H, 5-OH), 7.45 (m, 3H,
δ
=
181.8, 163.3, 158.5, 156.2, 151.8, 149.6, 131.6, 129.8, 127.8, 118.8, 116.3, 112.3, 104.8, 103.1, 99.3,
60.8, 48.6 (2 × C), 45.2 (2 × C) ppm. IR (KBr) ν 3494, 2842, 1654, 1605, 1583, 1491, 1452, 1353,
1255, 1179, 1107, 1026, 992, 799, 670, 549 cm-1.
5.1.19. 5, 7-Dihydroxy-8-methoxy-2-(3-(4-methylpiperazin-1-yl)phenyl)-4H-chromen-4-one (20c).
Yellow solid (55 mg, 58% yield). mp 265~268 oC. HRMS (ESI+) calcd for C21H23N2O5 [M + H]+:
1
383.1607, found 383.1599. H NMR (300 MHz, DMSO-d6):
δ = 12.52 (s, 1H, 5-OH), 10.89 (s, 1H,
7-OH), 7.52 (m, 3H, Ar-H), 7.27 (d, 1H, J= 7.50 Hz, Ar-H), 7.11 (s, 1H, CHCO), 6.34 (s, 1H, Ar-H),
3.85 (s, 3H, ArOCH3), 3.35 (s, 4H, Ar-N(CH2CH2)2N), 3.01 (s, 3H, NCH3), 2.73 (s, 4H,
Ar-N(CH2CH2)2N) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.1, 163.2, 157.4, 156.1, 150.1, 149.6,
131.8, 130.1, 127.7, 119.4, 117.3, 113.1, 105.1, 103.7, 99.1, 61.0, 52.1 (2 × C), 45.5 (2 × C), 42.2 ppm.
IR (KBr) ν 3433, 2944, 2679, 2606, 1658, 1592, 1522, 1434, 1364, 1275, 1257, 1026, 921, 837, 780,
692, 553 cm-1.
5.1.20. 2-(3-(1, 4-Diazepan-1-yl)phenyl)-5, 7-dihydroxy-8-methoxy-4H-chromen-4-one (20d).
o
Yellow solid (35 mg, 46.8% yield). mp 208~210 C. HRMS (ESI+) calcd for C21H22N2O5 [M +
H]+: 383.1529, found 383.1599.1H NMR (300 MHz, DMSO-d6):
δ = 12.55 (s, 1H, 5-OH), 7.31 (m, 3H,
Ar-H), 6.97 (s, 1H, Ar-H), 6.93 (s, 1H, CHCO), 6.23 (s, 1H, Ar-H), 3.83 (s, 3H, ArOCH3), 3.61 (s, 4H,
Ar-N(CH2)2), 2.95 (s, 2H, NHCH2), 2.72 (s, 2H, NHCH2), 1.86 (m, 2H, NCH2CH2CH2NH) ppm. 13C
NMR (75 MHz, DMSO-d6):
δ = 181.7, 163.5, 159.0, 156.3, 149.5, 148.5, 131.8, 130.0, 127.9, 114.9,
113.1, 108.4, 104.6, 102.9, 99.4, 60.7, 50.9, 47.3, 47.2, 46.9, 27.9 ppm. IR (KBr) ν 3410, 2927, 1652,
1598, 1499, 1368, 1175, 1107, 1026, 838, 772, 690, 561 cm-1.

ACCEPTED MANUSCRIPT
5.1.21. 7-Benzyloxy-2-(4-fluorophenyl)-5, 8-dihydroxy-4H-chromen-4-one (21).
Compound 16b (7.8 g, 17.7 mmol) was dissolved in DMF (55 mL) and K2CO3 (4.9 g, 35.5 mmol)
was added. Benzyl bromide (2.8 mL, 23 mmol) was added dropwise and then the mixture was heated
to 60 oC for 5 h. The reaction was monitored by TLC. After the end of the reaction, the mixture was
cooled to room temperature, filtered, and the cake was washed with methanol (20 mL). The filtrate was
adjusted to pH = 1 - 2 with 6 M HCl and the mixture was stirred at room temperature overnight to
precipitate a large amount of solid. Then the mixture was filtered and the cake was washed with
methanol (10 mL) and dried to give an Orange yellow solid (2.6 g, 38.8% yield). mp 290-292 oC. 1H
NMR (300 MHz, DMSO-d6):
δ = 12.69 (s, 1H, 5-OH), 9.13 (s, 1H, 8-OH), 7.92 (d, 2H, J= 9.00 Hz,
Ar-H), 7.45 (m, 5H, Ar-H), 7.12 (d, 2H, J= 9.00 Hz, Ar-H), 6.93 (s, 1H, CHCO), 6.79 (s, 1H, Ar-H),
5.34 (s, 2H, OCH2-Ar) ppm.
5.1.22. 7-Benzyloxy-2-(4-fluorophenyl)-5-hydroxy-8-methoxy-4H-chromen-4-one (22a).
Compound 21 (720 mg, 1.9 mmol) was dissolved in acetonitrile (60 mL) and K2CO3 (660 mg, 4.8
mmol) and dimethyl sulfate (240 mg, 2.1mmol) were added. The mixture was refluxed for 4.5 h and
the reaction was monitored by TLC. After the end of the reaction, the mixture was cooled to room
temperature, filtered, and the cake was washed with dichloromethane (20 mL). The filtrate was
evaporated under reduced pressure. The crude products were recrystallized from mixed solvent of
n-hexane and methylene chloride to give a yellow solid (685 mg, 92% yield). mp 210-212 oC. 1H NMR
(300 MHz, DMSO-d6):
δ = 12.70 (s, 1H, 5-OH), 7.93 (d, 2H, J= 9.00 Hz, Ar-H), 7.46 (m, 5H, Ar-H),
7.10 (d, 2H, J= 9.00 Hz, Ar-H), 6.94 (s, 1H, CHCO), 6.80 (s, 1H, Ar-H), 5.33 (s, 2H, OCH2-Ar), 3.85
(s, 3H, ArOCH3) ppm.
5.1.23. 7-Benzyloxy-2-(4-fluorophenyl)-5-hydroxy-8-isopropoxy-4H-chromen-4-one (22b).
Compound 21 (1 g, 2.65 mmol) was dissolved in DMF (15 mL) and K2CO3 (550 mg, 4.0 mmol)
and bromoisopropane (490 mg, 4.0 mmol) were added. The mixture was heated to 80 oC for 5 h and the
reaction was monitored by TLC. After the end of the reaction, the hot mixture was poured into 10
volumes of ice water to precipitate a large amount of solid. The mixture was filtered and the cake was
recrystallized from mixed solvent of n-hexane and methanol to give a yellow solid (580 mg, 52.3%
yield). mp 215-216 oC. 1H NMR (300 MHz, DMSO-d6):
δ = 12.89 (s, 1H, 5-OH), 7.88 (d, 2H, J= 9.00
Hz, Ar-H), 7.52 (m, 5H, Ar-H), 7.21 (d, 2H, J= 9.00 Hz, Ar-H), 6.94 (s, 1H, CHCO), 6.80 (s, 1H, Ar-H),
5.33 (s, 2H, OCH2-Ar), 4.45 (m, 1H, CH(CH3)2), 1.29 (d, 6H, J= 6.00 Hz, CH(CH3)2) ppm.
5.1.24. 7-Benzyloxy-8-cyclopentyloxy-2-(4-fluorophenyl)-5-hydroxy-4H-chromen-4-one (22c).
Compound 21 (1 g, 2.65 mmol) was dissolved in DMF (15 mL) and K2CO3 (550 mg, 4.0 mmol)
and bromoisopropane (490 mg, 4.0 mmol) were added in the presence of N2. The mixture was heated to
90 oC for 10 h and the reaction was monitored by TLC. After the end of the reaction, the hot mixture

ACCEPTED MANUSCRIPT
was poured into 10 volumes of ice water to precipitate a large amount of solid. The mixture was
filtered and the cake was purified by polyamide column chromatography to give a yellow solid (850
mg, 72% yield). mp 230-233 oC. 1H NMR (300 MHz, DMSO-d6):
δ =12.60 (s, 1H, 5-OH), 7.65 (d, 2H,
J= 9.00 Hz, Ar-H), 7.49 (m, 5H, Ar-H), 7.19 (d, 2H, J= 9.00 Hz, Ar-H), 6.98 (s, 1H, CHCO), 6.36 (s,
1H, Ar-H), 5.32 (s, 2H, OCH2-Ar), 4.86 (m, 1H, CH(CH2)4), 1.67 (m, 8H, CH(CH2)4) ppm.
5.1.25. 7-Benzyloxy-8-cyclohexyloxy-2-(4-fluorophenyl)-5-hydroxy-4H-chromen-4-one (22d).
Cyclohexanol (5 mL, 48 mmol) and triethylamine (13.3 mL, 96 mmol) were added in
dichloromethane (30 mL). Methanesulfonyl chloride (5.5 mL, 72 mmol, diluted with 10 mL of
o
dichloromethane) was added dropwise below 5 C and then the mixture was heated to room
temperature for 5 h. The reaction was monitored by TLC. After the end of the reaction, the mixture was
filtered and the filtrate was wash with 1 M HCl (20 mL), Saturated sodium bicarbonate solution (20 mL)
and Saturated saline solution (20 mL) and then dried by anhydrous sodium sulfate. The mixture was
filtered and the filtrate was evaporated under reduced pressure to give the oily compound of
methanesulfonyl cyclohexanol (1.06 g).
Compound 21 (1.5 g, 3.97 mmol) was dissolved in DMF (20 mL) and K2CO3 (1.37 g, 10 mmol)
and methanesulfonyl cyclohexanol (1.06 g) were added in the presence of N2. The mixture was heated
to 80 oC for 8 h and the reaction was monitored by TLC. After the end of the reaction, the hot mixture
was poured into 10 volumes of ice water and adjusted to pH = 4 - 5 with 1 M HCl to precipitate a large
amount of solid. The mixture was filtered and the cake was purified by polyamide column
chromatography to give a yellow solid (1.1 g, 63.2% yield). mp 235-238 oC. 1H NMR (300 MHz,
DMSO-d6):
δ = 12.91 (s, 1H, 5-OH), 7.78 (d, 2H, J= 9.00 Hz, Ar-H), 7.50 (m, 5H, Ar-H), 7.26 (d, 2H,
J= 9.00 Hz, Ar-H), 6.94 (s, 1H, CHCO), 6.77 (s, 1H, Ar-H), 5.30 (s, 2H, OCH2-Ar), 4.00 (m, 1H,
CH(CH2)5), 1.90 (m, 4H, CH(CH2)5), 1.53 (m, 2H, CH(CH2)5), 1.21 (m, 4H, CH(CH2)5) ppm.
The synthesis methods of 23a-23l were shown in supporting information.
5.1.26. 5, 7-Dihydroxy-8-methoxy-2-(4-(pyrrolidin-1-yl)phenyl)-4H-chromen-4-one (24a).
Compound 23a (168 mg, 0.38 mmol), Pd/C (40 mg) and tetrahydrofuran (10 mL) were added in
methanol (10 mL) in the presence of H2. The mixture was heated to 35 oC for 7 h and the reaction was
monitored by TLC. After the end of the reaction, the mixture was cooled to room temperature, filtered
and the filtrate was evaporated under reduced pressure. The crude product was purified over silica gel
o
column chromatography (# 100-200) to give a yellow solid (100 mg, 74.4% yield). mp 185-188 C.
1
HRMS (ESI+) calcd for C20H20NO5 [M + H]+: 354.1341, found 354.1274. H NMR (300 MHz,
DMSO-d6):
6.55 (s, 1H, CHCO), 6.46 (s, 1H, Ar-H), 4.00 (s, 3H, ArOCH3), 3.42 (s, 4H, N(CH2CH2)2), 1.70 (s, 4H,
N(CH2CH2)2) ppm. 13C NMR (75 MHz, DMSO-d6):
= 187.0, 168.8, 152.2, 150.9, 143.7, 140.4, 127.6,
δ = 12.77 (s, 1H, 5-OH), 7.86 (d, 2H, J= 9.00 Hz, Ar-H), 6.97 (d, 2H, J= 9.00 Hz, Ar-H),
δ

ACCEPTED MANUSCRIPT
127.1 (2 × C), 124.4, 113.0 (2 × C), 105.6, 98.6, 94.9, 56.6, 53.9 (2 × C), 23.1 (2 × C) ppm. IR (KBr) ν
3651, 2854, 1532, 1562, 1523, 1294, 1165, 1125, 1023, 1010, 863, 837, 701, 671 cm-1.
3.1.27. 2-(4-(Diethylamino)phenyl)-5, 7-dihydroxy-8-methoxy-4H-chromen-4-one (24b).
Compound 23b (180 mg). Yellow solid (75 mg, 53% yield). mp 165-170 oC. HRMS (ESI+) calcd
for C20H22NO5 [M + H]+: 356.1498, found 356.1480. 1H NMR (300 MHz, DMSO-d6):
δ = 12.81 (s, 1H,
5-OH), 10.63 (s, 1H, 7-OH), 7.88 (d, 2H, J= 9.00 Hz, Ar-H), 6.81 (d, 2H, J= 9.00 Hz, Ar-H), 6.70 (s,
1H, CHCO), 6.25 (s, 1H, Ar-H), 3.84 (s, 3H, ArOCH3), 3.44 (q, 4H, J= 6.00 Hz, N(CH2CH3)2), 1.14 (t,
6H, J= 6.00 Hz, N(CH2CH3)2) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.0, 164.7, 157.2, 156.7,
150.7, 149.8, 128.5 (2 × C), 128.0, 116.2, 111.7 (2 × C), 103.8, 101.1, 99.2, 61.5, 44.4 (2 × C), 12.9 (2
× C) ppm. IR (KBr) ν 3183, 2970, 1564, 1503, 1414, 1386, 1350, 1198, 1156, 1022, 944, 820, 712
cm-1.
5.1.28. 5, 7-Dihydroxy-8-methoxy-2-(4-morpholinophenyl)-4H-chromen-4-one (24c).
Compound 23c (145 mg). Yellow solid (55 mg, 47.4% yield). mp 190-195 oC. HRMS (ESI+) calcd
for C20H20NO6 [M + H]+: 370.1291, found 370.1271. 1H NMR (300 MHz, DMSO-d6):
δ = 12.77 (s, 1H,
5-OH), 10.68 (s, 1H, 7-OH), 7.85 (d, 2H, J= 9.00 Hz, Ar-H), 6.87 (d, 2H, J= 9.00 Hz, Ar-H), 6.54 (s,
1H, CHCO), 6.46 (s, 1H, Ar-H), 3.98 (s, 3H, ArOCH3), 3.77 (s, 4H, N(CH2CH2)2O), 3.23 (s, 4H,
N(CH2CH2)2O) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.0, 163.9, 157.1, 156.2, 151.5, 145.0,
127.6 (2 × C), 118.7, 116.1, 113.2 (2 × C), 105.0, 103.7, 99.0, 66.0, 61.0 (2 × C), 48.0 (2 × C) ppm. IR
(KBr) ν 3245, 2829, 1562, 1524, 1493, 1327, 1265, 1212, 1123, 1023, 852, 814, 752, 663 cm-1.
5.1.41. 5, 7-Dihydroxy-8-methoxy-2-(4-(piperazin-1-yl)phenyl)-4H-chromen-4-one (24d).
Compound 23d (178 mg). Yellow solid (60 mg, 42% yield). mp 190-195 oC. HRMS (ESI+) calcd
1
for C20H21N2O5 [M + H]+: 369.1450, found 369.1439. H NMR (300 MHz, DMSO-d6):
δ = 12.56 (s,
1H, 5-OH), 7.89 (d, 2H, J= 9.00 Hz, Ar-H), 7.14 (d, 2H, J= 9.00 Hz, Ar-H), 6.82 (s, 1H, CHCO), 6.35
(s, 1H, Ar-H), 3.98 (s, 3H, ArOCH3), 3.20 (s, 4H, N(CH2CH2)2NH), 2.90 (s, 4H, N(CH2CH2)2NH) ppm.
13C NMR (75 MHz, DMSO-d6):
δ = 182.1, 163.3, 158.5, 156.2, 151.8, 149.6, 127.8 (2 × C), 118.8,
116.3, 113.3 (2 × C), 104.8, 103.1, 99.3, 60.8, 48.6 (2 × C), 45.2 (2 × C) ppm. IR (KBr) ν 3537, 2953,
1643, 1605, 1575, 1482, 1443, 1386, 1215, 1186, 1124, 992, 837, 670, 553 cm-1.
5.1.29. 5, 7-Dihydroxy-8-methoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-4H-chromen-4-one (24e).
Compound 23e (165 mg). Yellow solid (50 mg, 37.4% yield). mp 190-195 oC. HRMS (ESI+) calcd
1
for C21H23N2O5 [M + H]+: 383.1607, found 383.1640. H NMR (300 MHz, DMSO-d6):
δ = 12.70 (s,
1H, 5-OH), 7.82 (d, 2H, J= 9.00 Hz, Ar-H), 6.99 (d, 2H, J= 9.00 Hz, Ar-H), 6.57 (s, 1H, CHCO), 6.43
(s, 1H, Ar-H), 4.05 (s, 3H, ArOCH3), 3.44 (t, J= 6.00 Hz, 4H, Ar-N(CH2CH2)2N), 3.01 (t, J= 6.00 Hz,
4H, Ar-N(CH2CH2)2N), 2.43 (s, 3H, NCH3) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.3, 167.8,
157.5, 156.1, 150.9, 149.6, 127.3 (2 × C), 118.5, 116.4, 113.0 (2 × C), 105.1, 103.7, 99.1, 61.0, 52.1 (2

ACCEPTED MANUSCRIPT
× C), 45.5 (2 × C), 42.2 ppm. IR (KBr) ν 3430, 2854, 2648, 1653, 1454, 1367, 1228, 1210, 1026, 921,
837, 780, 692, 583 cm-1.
5.1.30. 2-(4-(1, 4-Diazepan-1-yl)phenyl)-5, 7-dihydroxy-8-methoxy-4H-chromen-4-one (24f).
Compound 23f (165 mg). Yellow solid (50 mg, 37.4% yield). mp 202-205ºC. HRMS (ESI+) calcd
1
for C21H23N2O5 [M + H]+: 383.1607, found 383.1599. H NMR (300 MHz, DMSO-d6):
δ = 12.80 (s,
1H, 5-OH), 7.88 (d, 2H, J= 9.00 Hz, Ar-H), 6.88 (d, 2H, J= 9.00 Hz, Ar-H), 6.72 (s, 1H, CHCO), 6.27
(s, 1H, Ar-H), 3.84 (s, 3H, ArOCH3), 3.63 (s, 4H, Ar-N(CH2)2), 2.98 (s, 2H, NHCH2), 2.73 (s, 2H,
NHCH2), 1.86 (m, 2H, NCH2CH2CH2NH) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 181.5, 163.6,
157.5, 156.3, 153.4, 149.3, 130.6, 127.9, 125.0, 119.7, 111.7, 111.2, 103.1, 100.2, 98.8, 60.2, 58.2, 51.6,
48.2, 47.0, 28.9 ppm. IR (KBr) ν 3131, 2854, 1655, 1578, 1400, 1358, 1101, 872, 775, 690, 550 cm-1.
5.1.31. 5, 7-Dihydroxy-8-isopropoxy-2-(4-(piperazin-1-yl)phenyl)-4H-chromen-4-one (24g).
Compound 23g (180 mg). Yellow solid (80 mg, 54.6% yield). mp 210-213 oC. HRMS (ESI+) calcd
1
for C22H25N2O5 [M + H]+: 397.1763, found 397.1710. H NMR (300 MHz, DMSO-d6):
δ = 12.75 (s,
1H, 5-OH), 7.90 (d, 2H, J= 9.00 Hz, Ar-H), 7.08 (d, 2H, J= 9.00 Hz, Ar-H), 6.76 (s, 1H, CHCO), 6.27
(s, 1H, Ar-H), 4.38 (m, 1H, CH(CH3)2), 3.28 (s, 4H, N(CH2CH2)2NH), 2.84 (s, 4H, N(CH2CH2)2NH),
1.30 (d, 6H, J= 6.09 Hz, CH(CH3)2) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.0, 164.4, 156.3,
152.1, 151.1, 149.8, 127.6 (2 × C), 125.1, 119.0, 113.1 (2 × C), 103.4, 101.7, 98.8, 73.8, 50.7 (2 × C),
46.6 (2 × C), 22.3 (2 × C) ppm. IR (KBr) ν 3379, 2813, 1616, 1525, 1506, 1368, 1262, 1237, 1119,
1026, 821, 663, 595 cm-1.
5.1.32. 5, 7-Dihydroxy-8-isopropoxy-2-(4-(4-methylpiperazin-1-yl)phenyl)-4H-chromen-4-one (24h).
Compound 23h (155 mg). Yellow solid (65 mg, 51.2% yield). mp 240-242 oC. HRMS (ESI+)
calcd for C23H27N2O5 [M + H]+: 411.1920, found 411.1890. 1H NMR (300 MHz, DMSO-d6):
δ = 12.76
(s, 1H, 5-OH), 10.59 (s, 1H, 7-OH), 7.92 (d, 2H, J= 9.00 Hz, Ar-H), 7.11 (d, 2H, J= 9.00 Hz, Ar-H),
6.79 (s, 1H, CHCO), 6.28 (s, 1H, Ar-H), 4.37 (m, 1H, CH(CH3)2), 3.34 (s, 4H, Ar-N(CH2CH2)2N), 3.34
(s, 3H, NCH3), 2.45 (s, 4H, Ar-N(CH2CH2)2N), 1.30 (d, 6H, J= 6.09 Hz, CH(CH3)2) ppm. 13C NMR
(75 MHz, DMSO-d6):
δ = 181.0, 167.8, 157.5, 156.1, 150.9, 149.6, 127.3 (2 × C), 118.5, 116.4, 113.0
(2 × C), 105.1, 103.7, 99.1, 74.0, 52.1 (2 × C), 45.5 (2 × C), 42.2, 22.1 (2 × C) ppm. IR (KBr) ν 3430,
2854, 2648, 1653, 1454, 1367, 1228, 1210, 1026, 921, 837, 780, 692, 583 cm-1.
5.1.33. 5, 7-Dihydroxy-8-cyclopentyloxy-2-(4-(piperazin-1-yl)phenyl)-4H-chromen-4-one (24i).
Compound 23i (160 mg). Yellow solid (65 mg, 49.3% yield). mp 255-258 oC. HRMS (ESI+) calcd
1
for C24H27N2O5 [M + H]+: 423.1920, found 423.1913. H NMR (300 MHz, DMSO-d6):
δ = 12.75 (s,
1H, 5-OH), 7.88 (d, 2H, J= 9.00 Hz, Ar-H), 7.04 (d, 2H, J= 9.00 Hz, Ar-H), 6.76 (s, 1H, CHCO), 6.26
(s, 1H, Ar-H), 4.81 (m, 1H, CH(CH2)4), 3.40 (s, 4H, N(CH2CH2)2NH), 2.83 (s, 4H, N(CH2CH2)2NH),
1.50 (m, 8H, CH(CH2)4) ppm. 13C NMR (75 MHz, DMSO-d6):
δ = 182.0, 164.6, 155.8, 152.1, 151.1,